The mitochondrial Ca 2؉ -independent phospholipase A 2 is activated during energy-dependent Ca 2؉ accumulation under conditions where there is a sustained depression of the membrane potential. This activation is not dependent on induction of the mitochondrial permeability transition. Bromoenol lactone, which inhibits the phospholipase, is effective as an inhibitor of the transition, and this action can be overcome by low levels of exogenous free fatty acids. Apparently, activation of the Ca 2؉ -independent phospholipase is a factor in the mechanisms by which depolarization and Ca 2؉ accumulation promote opening of the permeability transition pore. Sustained activity of the Ca 2؉ -independent phospholipase A 2 promotes rupture of the outer mitochondrial membrane and spontaneous release of cytochrome c on a time scale similar to that of apoptosis occurring in cells. However, more swelling of the matrix space must occur to provoke release of a given cytochrome c fraction when the enzyme is active, compared with when it is inhibited. Through its effects on the permeability transition and release of intermembrane space proteins, the mitochondrial Ca 2؉ -independent phospholipase A 2 may be an important factor governing cell death caused by necrosis or apoptosis.Mitochondria from rat liver and rabbit heart have been shown to contain a Ca 2ϩ -independent phospholipase A 2 (iPLA 2 ) 3 that has a molecular mass of ϳ80 kDa (1, 2). Like phospholipases of this type from other sources (3, 4), the mitochondrial enzyme is inactivated by bromoenol lactone (BEL), which acts through an activity-dependent mechanism, leading to a covalent modification within the active site (5). No physiological function of the iPLA 2 in mitochondria has been established, but it has been shown that pretreatment with BEL attenuates the loss of phospholipids that accompanies ischemia/reperfusion injury and reduces the size of infarcts by ϳ50% (2).The relationship between mitochondrial energetic status and iPLA 2 activity is an important factor to consider when contemplating potential physiological and pathophysiological roles of the enzyme. More specifically, activity is not seen in mitochondria that are respiring under state 4 conditions but is manifest upon the addition of uncoupler and is fully manifest following the development of inner membrane pores (1). The former property suggests that transient periods of deenergization might cause a transient activation of the iPLA 2 in vivo, with a resulting accumulation of free fatty acids in mitochondria. Such an accumulation could be of interest in many regards, including opening of the permeability transition pore, which is favored by low levels of these compounds (6 -8). Occurrence of the permeability transition leads to apoptosis in many cell types (9 -11), so scenarios arise in which the iPLA 2 contributes to the control of apoptosis by influencing the permeability transition and wherein the facilitative effects of depolarization on the transition (12, 13) might occur through activity of this enzyme...

The production of cytokines by human conjunctival epithelial cells following stimulation was investigated. Primary cultures of human conjunctival epithelial cells were characterized by morphology and keratin expression. Cultured epithelial cells were treated with varying concentrations of lipopolysaccharide, interleukin (IL)-1 beta, calcium ionophore A23187, or phorbol myristate acetate, and cytokine secretion was determined over specified intervals. Culture supernatants and cell lysates were analyzed by ELISA for IL-1 beta, IL-3, IL-4, IL-5, IL-6, IL-8, IL-11, IL-1 receptor antagonist (IL-1ra), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony stimulating factor (GM-CSF). With the exception of IL-1ra, unstimulated conjunctival epithelial cells produced cytokines at relatively low or undetectable levels. IL-1ra was detected in both culture supernatants and cell lysates under basal conditions. In response to stimuli, conjunctival epithelial cells secreted the proinflammatory cytokines TNF-alpha, IL-6, IL-8, and GM-CSF in a dose- and time-dependent fashion. After stimulation, the intracellular levels of IL-1ra increased in these cells but the supernatant-associated levels remained unchanged. None of the other cytokines evaluated (IL-1 beta, IL-3, IL-4, IL-5, and IL-11) were detected in supernatants or lysates of resting or stimulated cells. These findings suggest that conjunctival epithelial cells may contribute to the pathogenesis of human ocular diseases by production of proinflammatory cytokines. Further evaluation of these cells as targets of therapy is warranted.

Olopatadine (AL-4943A; KW-4679) [(z)-11-[3-(dimethylamino)propylidene]-6, 11-dihydrodibenz[b,e]oxepine-2 acetic acid hydrochloride] is an anti-allergic agent which inhibits mast cell mediator release and possesses histamine H1 receptor antagonist activity. Studies were conducted to characterize the in vitro and in vivo pharmacological profile of this drug relevant to its topical ocular use. AL-4943A inhibits histamine release in a concentration-dependent fashion (IC50 = 559 microM) from human conjunctival mast cell preparations in vitro. Histamine release was not stimulated by AL-4943A at concentrations as high as 10 mM. In contrast, ketotifen stimulated histamine release at concentrations slightly higher than effective inhibitory concentrations. AL-4943A did not display any in vitro cyclooxygenase or 5-lipoxygenase inhibition. Topical ocular application of AL-4943A effectively inhibits antigen- and histamine-stimulated conjunctivitis in guinea pigs. Passive anaphylaxis in guinea pig conjunctiva was attenuated by AL-4943A applied 30 min prior to intravenous or topical ocular antigen challenge (ED50 values 0.0067% and 0.0170%, w/v, respectively). Antihistaminic activity in vivo was demonstrated using a model of histamine-induced vascular permeability in guinea pig conjunctiva. AL-4943A applied topically from 5 min to 24 hrs prior to histamine challenge effectively and concentration-dependently inhibited the vascular permeability response, indicating the compound has an acceptable onset and a long duration of action. Drug concentrations 5-fold greater than those effective against histamine-stimulated conjunctival responses failed to inhibit vascular permeability responses induced with either serotonin or Platelet-Activating-Factor. These data indicate that the anti-histaminic effect observed with AL-4943A is specific. These anti-allergic/antihistaminic activities of AL-4943A observed in preclinical model systems have been confirmed in clinical trials in allergic patients.

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Mass spectra of several triglycerides of specific structure or with specific deuterium labeling have been measured with a low resolution mass spectrometer. With saturated triglycerides the abundances of ions characteristic of the component acids, [M-RCO 2 ] § increase with increasing chain length, and [M-RCO2CH2]+ decrease with increasing chain length. Unsaturation in the acyl moiety causes the abundant formation of [RCO-1] +. Structures have been suggested for a number of the main peaks obtained from saturated triglycerides, and high resolution spectra of one triglyceride agree with the postulated structures. The peaks, [RCO+74] § [RCO+115] + and [RCO+128+14n]+, represent structures which contain the glyceryl portion of the triglyceride, since in case of the replacement of its hydrogens with deuteriums, these peaks are shifted accordingly. Evidence which indicates the possibility of determining the location of unsaturation by the interruption of homology of the [RCO+128+14n] § series, brought about by the addition of deuterium to the unsaturated linkages, is introduced.Further evidence is also presented, which indicates that the [M-RCO2CH2] + ions arise from the positions 1 and 3 and, in agreement with earlier studies from other laboratories, it is thus possible to identify the acyl groups attached to the 1 and 2 positions of the glyceryl moiety.